PROJECT SUMMARY Pseudomonas aeruginosa is an opportunistic pathogen found in acute infections (burns, wounds, ventilator associated pneumonia, eye infections) and chronic infections of the foot (diabetic ulcers) and lung (cystic fibrosis). This bacterium commonly survives in these contexts as a biofilm, the formation and high-level antibiotic tolerance of which interferes with effective patient treatment. A defining aspect of P. aeruginosa is its ability to make phenazines, colorful redox-active pigments that mediate a variety of processes, including survival within the anoxic interior of biofilms. Like biofilms, mucus collecting in the lungs of CF patients exhibits steep oxygen (O2) gradients. Over time, P. aeruginosa commonly dominates the microbial population in the CF lung, as its physiology permits it to thrive in this environment. As O2 declines, phenazines rise, and the concentration of certain phenazines, such as pyocyanin (PYO)?a virulence factor in animal infection models? is correlated with declining lung function. While how PYO is made and impacts diverse cell types (positively for the producer, and negatively for the host) is well understood, the potential impact of reducing PYO concentration for host-pathogen interactions is unknown. Recently, we discovered a novel PYO demethylase (PodA) made by members of the Mycobacterium fortuitum complex, which can infect CF patients. PodA converts PYO to 1-hydroxy-phenazine (1OHPHZ), and blocks biofilm formation and development. PYO is known to trigger eDNA release and promote biofilm formation, as well as sustain P. aeruginosa's anaerobic metabolism via a process called extracellular electron transfer (EET). In infections where PYO is abundant, we hypothesize that PodA might help control P. aeruginosa by inhibiting eDNA release and abrogating EET by converting PYO to 1OHPHZ. Here, we seek to gain a fundamental scientific understanding of PodA and its mechanism of anti-biofilm activity as a first step towards evaluating its therapeutic potential. First, how does PodA catalyze PYO demethylation? Second, what is the consequence of PYO removal and 1OHPHZ formation for P. aeruginosa? Third, might PodA activity potentiate the effectiveness of conventional antibiotics in controlling P. aeruginosa in slowly-growing, O2-limited biofilm regions? To answer these questions, we propose two specific aims. Aim 1 will explore the enzymatic activity and mechanism of action of the PodA enzyme in detail. Aim 2 will probe the mechanisms underpinning PodA's inhibition of P. aeruginosa biofilm development at early and late stages, and whether it can sensitize P. aeruginosa to tobramycin and ciprofloxacin. Attainment of these objectives will lay the foundation of basic knowledge necessary to evaluate the potential usage of PodA as a therapeutic enzyme.